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PubMed1993

A simple procedure for crystallization of the Schiff reagent.

Formation of crystals in Schiff reagents prepared from SO2 gas previously has been reported either soon after preparation, using high dye concentrations and heating, or after long periods of storage at room temperature. With the first type of procedure only a low yield of crystals accompanied by dye precipitation was obtained. Crystallization without dye precipitation took place if the reagent, prepared with pararosaniline base or chloride in a saturated SO2 solution, was stored for a sufficient time at room temperature in partly filled flasks. These crystals remained colorless if washed with acid alcohol after being separated by filtration. Schiff reagents layered with paraffin oil or supplemented with 0.1 M hydroquinone took much longer to crystallize, suggesting that crystallization is promoted by the partial oxidation of sulfurous acid to sulfuric acid. A high yield of crystals can be obtained at room temperature after as little as 24 hr by adding 0.04 M of H2SO4 to a Schiff reagent prepared with 2% pararosaniline chloride in a saturated SO2 solution. A Schiff reagent prepared with only 0.2% of these crystals gives an intense staining in the Feulgen and in the Periodic acid-Schiff reactions.

Animals↗
PubMedDate not supplied

The effect of flowable resin composite on microleakage in class V cavities.

This in vitro study investigated the microleakage of flowable resin composite as a restorative material and as a liner (either light cured separately or co-cured with hybrid resin composite) in Class V cavities. A light-cured hybrid resin composite was used as a control. Twenty extracted human premolars were prepared with standardized Class V cavity outlines on the buccal and lingual surfaces. The occlusal margin of the cavities was on enamel and the gingival margin was on dentin. One bottle adhesive system (Single Bond) was used after etching enamel and dentin with 34.5% phosphoric acid for 15 seconds. The cavities were randomly divided into four groups of 10 each and restored according to the manufacturers' instructions: Group I-Hybrid resin composite (Z100); Group II-Flowable resin composite (Filtek Flow); Group III-Flowable resin composite (Filtek Flow)+Hybrid resin composite (Z100); light cured separately; Group IV-Flowable resin composite (Filtek Flow)+Hybrid resin composite (Z100); co-cured. The samples were thermocycled 200 times with a 30-second dwell time. They were then immersed in a 0.5% basic fuchsin solution for 24 hours, sectioned and analyzed by stereomicroscopy. The degree of dye penetration was recorded and analyzed with the Kruskal-Wallis and Mann-Whitney U tests. The results of this study indicate that there was no leakage at the occlusal margin for either restoration. Statistically significant differences were found among the groups at the gingival margin. No statistically significant difference was observed between the occlusal and gingival margins except in Group IV. The combination of flowable resin composite and hybrid composite light cured separately yielded the best result in this study. The most leakage was observed when this combination was co-cured. The resistance to microleakage of flowable resin composite as a restorative material is similar to that of hybrid resin composite.

Acid Etching, Dental↗
PubMed2001

Microleakage of composite restorations after acid or Er-YAG laser cavity treatments.

OBJECTIVES: The purpose of this study was to compare microleakage of Class V restorations following acid, laser or laser and acid treatment of cavity walls. METHODS: Standardized lingual and buccal Class V preparations were made in 18 human extracted third molars. The preparations were randomly assigned to three equal groups (n=12). Group 1: cavities were treated with 35% phosphoric acid. Group 2: cavities were irradiated with an Er-YAG laser at 2Hz and 250mJ on dentin and 300mJ on enamel, with water cooling. Group 3: cavities were irradiated with the laser before acid etching. Scotchbond 1 Adhesive System and Z100 resin composite were used for restorations. The specimens were stored in water for 24h at 37 degrees C and thermally cycled (500x) between 6-60 degrees C. After 24h immersion in 0.5% basic fuchsin, dye penetration was recorded according to an ordinal scale. Data were analysed using non-parametric statistical tests (Kruskal-Wallis and Mann-Whitney). RESULTS: On the occlusal walls, microleakage in acid etched cavities was significantly lower than that achieved after laser treatment (P<0.001) or after both treatments (P<0.05). On the gingival walls, no statistical differences were found. SIGNIFICANCE: Laser irradiation of enamel is not a valid alternative to acid-etching pretreatment for resin composite materials adhesion. Acid etching alone gave the lowest microleakage at the occlusal margin. No differences were found for microleakage on gingival wall, although lased dentin surfaces presented several characteristics that appear to be advantageous for bonding.

Acid Etching, Dental↗
PubMed2006

[Influence of vital dyes on the function of the outer blood-retinal barrier in vitro].

BACKGROUND: The use of vital dyes in "chromovitrectomy" allows the easier removal of less recognizable structures like epiretinal membranes (EM) or the inner limiting membrane (ILM). In recent years numerous studies demonstrated the use of indocyanine green (ICG), trypan blue (TB) and patent blue (PB) for this indication. Reports of the possible risk of these dyes, i. e. especially ICG, in respect of reduced visual acuity results, possible visual field defects or alterations of the retinal pigment epithelium (RPE) resulted in limitations in their application. MATERIAL AND METHODS: Human RPE cells and choroidal endothelial cells were cultured in monolayers on semipermeable membranes representing an in vitro model of the outer blood-retinal barrier. By measurement of the transepithelial electrical resistance (TER) the stable barrier function was determined. Two different models representing an air-filled and a fluid-filled eye were tested on the one hand by addition of the dye to the culture medium and, on the other, by direct application on the cell monolayer. In these two models ICG (5 mg/ml, 0.5 mg/ml, 0.125 mg/ml), TB (1.5 mg/ml, 0.15 mg/ml) and PB (2.4 mg/ml, 0.24 mg/ml) were applied for three minutes and the influence on the barrier function was determined. RPE cell growth was also tested in these two models after the application of ICG, TB and PB. Finally, monolayers of RPE cells were evaluated by transmission electron microscopy (TEM). RESULTS: After application of TB, PB and the lowest concentration of ICG of 0.125 mg/ml, the TER remained stable in both models. In contrast, ICG in a concentration of 5 mg/ml and 0.5 mg/ml caused a significant TER decrease in the model of the air-filled eye, whereas no influence on the function of the outer blood-retinal barrier was noted in the model of the fluid-filled eye. RPE cell growth rates were not influenced by the addition of the vital dyes, with the exception of ICG in a concentration of 5 mg/ml in the model of the air-filled eye, resulting in a temporary reduction of the cell count. In good correspondence to these results also in TEM intercellular blisters were noted after application of 5 mg/mL ICG for 3 minutes in the model of the air-filled eye. However, damage to the RPE cells themselves was not obvious. No pathological changes in the TEM were noted after application of TB and PB. CONCLUSIONS: The use of PB and TB at the posterior eye segment seems to be safe concerning damage to the PRE and its barrier function. In contrast, ICG in higher concentrations and with longer application times may cause a toxic effect on RPE morphology and function.

Blood-Retinal Barrier↗
PubMedDate not supplied

Allergic reactions to isosulfan blue in sentinel lymph node mapping.

BACKGROUND: Sentinel lymph node (SLN) biopsy is often used in the assessment of lymph node status in melanoma and early stage breast cancer. With the rapidly increasing use of the technique, we can now better characterize and assess the rate of adverse reactions to the dye. METHODS: A retrospective review of all patients undergoing SLN mapping at the Columbia-Presbyterian Breast Center were identified from June 2000 to July 2002. All patients who experienced allergic reactions were documented and records examined. RESULTS: In total, three out of 351 patients had allergic complications from the procedure. All three patients developed "blue hives" after injection with isosulfan blue. The incidence at our Breast Center was 0.9%. All were treated with intravenous corticosteroids and diphenhydramine and recovered within twenty-four hours. CONCLUSIONS: The increasing utilization of the sentinel lymph node technique will make these complications more common. A high index of suspicion and appropriate clinical management are recommended to minimize the potential morbidity of these reactions.

Breast Neoplasms↗
PubMed1996

Evaluation of an automated pyrogallol red-molybdate method for the measurement of urinary protein in rats.

Methods for quantitating urinary protein differ in their ranges of linearity, technical ease of performance, and applicability to automated analyzers. The Coomassie Brilliant Blue method is widely used but has limited linearity and its tendency to stain glassware has limited its application to automated analyzers. We evaluated a pyrogallol red-molybdate protein dye-binding method (Biotrol USA, Inc.) on a Hitachi 705 analyzer for the quantitation of urinary protein in rats. This method showed a wide range of linearity (up to 2.6 g/l) and good precision. Within-run CVs of 6.6% and 1.3% and between-day CVs of 10.9% and 1.1% were observed at mean protein concentrations of 0.16 g/l and 1.96 g/l, respectively. In addition, rat urine protein results from this method correlated well (r2 = 0.998, n = 40) with a Coomassie Brilliant Blue method (QuanTtest Blue, Quantimetrix Corporation). No significant or unexpected interferences were encountered with this method. We conclude that the automated pyrogallol red-molybdate method is an acceptable and practical alternative to the Coomassie Brilliant Blue method for the quantitation of urine protein in rats.

Animals↗
PubMed2004

Avidin/biotin-liposome system injected in the pleural space for drug delivery to mediastinal lymph nodes.

The objective of this study was to develop a more effective liposome-based method for delivering drugs to mediastinal nodes. Nodal uptake was determined after intrapleural injection of the avidin/biotin-liposome system in normal rats. The effect of injection sequence (avidin injected 2 h before biotin-liposomes and vice versa), volume injected, and administered dose of the agents is described. Pharmacokinetics of the avidin/biotin-liposome system was monitored with scintigraphic imaging by labeling the biotin-liposomes with technetium-99m ((99m)Tc). To identify the nodes during the biodistribution studies, patent blue dye was encapsulated in the biotin-liposomes. Tissue biodistribution studies were performed 22 h after injection of the (99m)Tc-blue-biotin-liposomes. When avidin was injected before (99m)Tc-blue-biotin-liposomes, better mediastinal node targeting (15.7%; p < 0.05) was achieved than when biotin-liposomes were injected first (8.3%) or when only biotin-liposomes were injected (1.0%). Injection of a small dose of liposomes (0.5 mg phospholipid) and avidin (0.5 mg) resulted in the most favorable drug delivery to mediastinal nodes and other organs. Intrapleural injection of the avidin/biotin-liposome system could potentially be used for drug delivery to disease processes such as lung cancer, anthrax, and tuberculosis that invade mediastinal nodes and use them as centers of incubation and dissemination.

Animals↗
PubMed2004

Recognition of planar and nonplanar ligands in the malachite green-RNA aptamer complex.

Ribonucleic acids are an attractive drug target owing to their central role in many pathological processes. Notwithstanding this potential, RNA has only rarely been successfully targeted with novel drugs. The difficulty of targeting RNA is at least in part due to the unusual mode of binding found in most small-molecule-RNA complexes: the ligand binding pocket of the RNA is largely unstructured in the absence of ligand and forms a defined structure only with the ligand acting as scaffold for folding. Moreover, electrostatic interactions between RNA and ligand can also induce significant changes in the ligand structure due to the polyanionic nature of the RNA. Aptamers are ideal model systems to study these kinds of interactions owing to their small size and the ease with which they can be evolved to recognize a large variety of different ligands. Here we present the solution structure of an RNA aptamer that binds triphenyl dyes in complex with malachite green and compare it with a previously determined crystal structure of a complex formed with tetramethylrosamine. The structures illustrate how the same RNA binding pocket can adapt to accommodate both planar and nonplanar ligands. Binding studies with single- and double-substitution mutant aptamers are used to correlate three-dimensional structure with complex stability. The two RNA-ligand complex structures allow a discussion of structural changes that have been observed in the ligand in the context of the overall complex structure. Base pairing and stacking interactions within the RNA fold the phosphate backbone into a structure that results in an asymmetric charge distribution within the binding pocket that forces the ligand to adapt through a redistribution of the positive partial charge.

Coloring Agents↗
PubMed1980

Recovery of pure ribosomal proteins from stained gels. A fast method of purification of active proteins.

A simple technique has been developed for eluting ribosomal proteins from stained gels in the presence of an acetic acid solution. The ribosomal proteins are then separated from the dye by anion-exchange chromatography under dissociating conditions. Ribosomal proteins purified by these methods give total cross-reaction with proteins obtained by standard procedures, when tested by immunodiffusion against their corresponding antibodies, and show the same electrophoretic mobility as standard proteins in bidimensional polyacrylamide gel systems. Ribosomal proteins L7/L12, recovered from stained gels and purified by these methods, are able to reconstitute the elongation-factor-G-dependent GTPase activity of ribosomal particles deprived of these proteins. Radioactive protein L1, recovered in the same way, is incorporated into a total reconstituted 50-S subunit, competing with an excess of standard L1 present in the pool of total proteins from 50-S subunits used for reconstitution. These results suggest that bidimensional electrophoresis can be considered an alternative system of purification of active proteins from complex mixtures.

Chromatography, Ion Exchange↗
PubMed2004

The application of sentinel node radiolocalization to solid tumors of the head and neck: a 10-year experience.

OBJECTIVES/HYPOTHESIS: The goals of the research study were to develop an easily mastered, accurate, minimally invasive technique of sentinel node radiolocalization with biopsy (SNRLB) in the feline model; to compare it with blue-dye mapping techniques; and to test the applicability of sentinel node radiolocalization biopsy in three head and neck tumor types: N0 malignant melanoma, N0 Merkel cell carcinoma, and N0 squamous cell carcinoma. STUDY DESIGN: Prospective consecutive series studies were performed in the feline model and in three head and neck tumor types: N0 malignant melanoma (43 patients), N0 Merkel cell carcinoma (8 patients), and N0 squamous cell carcinoma (20 patients). METHODS: The technique of sentinel node radiolocalization with biopsy was analyzed in eight felines and compared with blue-dye mapping. Patterns of sentinel node gamma emissions were recorded. Localization success rates were determined for blue dye and sentinel node with radiolocalization biopsy. In the human studies, all patients had sentinel node radiolocalization biopsy performed in a similar manner. On the morning of surgery, each patient had sentinel node radiolocalization biopsy of the sentinel lymph node performed using an intradermal or peritumoral injection of technetium Tc 99m sulfur colloid. Sentinel nodes were localized on the skin surface using a handheld gamma detector. Gamma count measurements were obtained for the following: 1) the "hot" spot/node in vivo before incision, 2) the hot spot/node in vivo during dissection, 3) the hot spot/node ex vivo, 4) the lymphatic bed after hot spot/node removal, and 5) the background in the operating room. The first draining lymph node(s) was identified, and biopsy of the node was performed. The radioactive sentinel lymph node(s) was submitted separately for routine histopathological evaluation. Preoperative lymphoscintigrams were performed in patients with melanoma and patients with Merkel cell carcinoma. In patients with head and neck squamous cell carcinoma, the relationship between the sentinel node and the remaining lymphatic basin was studied and all patients received complete neck dissections. The accuracy of sentinel node radiolocalization with biopsy, the micrometastatic rate, the false-negative rate, and long-term recurrence rates were reported for each of the head and neck tumor types. In the melanoma study, the success of sentinel node localization was compared for sentinel node radiolocalization biopsy, blue-dye mapping, and lymphoscintigraphy. In the Merkel cell carcinoma study, localization rates were evaluated for sentinel node radiolocalization biopsy and lymphoscintigraphy. In the head and neck squamous cell carcinoma study, the localization rate of sentinel node radiolocalization biopsy and the predictive value of the sentinel node relative to the remaining lymphatic bed were determined. All results were analyzed statistically. RESULTS: Across the different head and neck tumor types studied, sentinel node radiolocalization biopsy had a success rate approaching 95%. Sentinel node radiolocalization biopsy was more successful than blue-dye mapping or lymphoscintigraphy at identifying the sentinel node, although all three techniques were complementary. There was no instance of a sentinel node-negative patient developing regional lymphatic recurrence. In the head and neck squamous cell carcinoma study, there was no instance in which the sentinel node was negative and the remaining lymphadenectomy specimen was positive. CONCLUSION: In head and neck tumors that spread via the lymphatics, it appears that sentinel node radiolocalization biopsy can be performed with a high success rate. This technique has a low false-negative rate and can be performed through a small incision. In head and neck squamous cell carcinoma, the histological appearance of the sentinel node does appear to reflect the regional nodal status of the patient.

Adult↗
PubMedDate not supplied

Gas chromatographic/mass spectrometric confirmation of leucomalachite green in catfish (Ictalurus punctatus) tissue.

A gas chromatographic/mass spectrometric (GC/MS) method was developed to confirm the presence of leucomalachite green (LMG), a metabolite of the triphenylmethane dye malachite green (MG), in catfish tissue. Residues were isolated according to a previously described liquid chromatographic (LC)/VIS spectrometric analysis of MG and LMG in fish. In our isolation procedure, analytes are extracted from tissue with acetonitrile-buffer, partitioned into CH2Cl2, and applied to neutral alumina and propylsulfonic acid solid-phase extraction cartridges. Before GC/MS analysis, extracts prepared for the LC determinative method are eluted from a cyano solid-phase extraction cartridge, extracted into organic solvent, and concentrated for GC/MS analysis. Selected ion monitoring was performed by using 5 diagnostic ions (m/z 330, 329, 253, 210, and 165) of LMG. The method was validated by confirming LMG in tissue fortified with mixtures of MG and LMG (5 and 10 ng/g each) and in tissue from fish that had been exposed to low levels of MG.

Aniline Compounds↗
PubMed1996

Differentiation of low grade non-Hodgkin's lymphoma by digital image processing.

OBJECTIVE: To identify different types of low grade B-cell non-Hodgkin's lymphoma (NHL) classified according to the Revised European American Lymphoma and Kiel classification systems by means of digital image processing. STUDY DESIGN: Seventy-four touch imprints were scanned and analyzed. To compare common but intricate DNA stain with a routinely used panoptical dye, all lymphoma specimens had been stained by the Romanowsky-Giemsa method and 48 touch imprints redyed with Feulgen-Azure A. In both cases 30 features derived from size, and chromatin texture of each nucleus were evaluated. RESULTS: Feulgen-stained touch imprints showed a 59% average probability of correct identification. The division of mantle cell lymphoma and chronic lymphocytic leukemia was difficult. In contrast, it was possible to distinguish all different types of lymphomas investigated if Romanowsky-Giemsa stain was used. Correct diagnoses were achieved for mantle cell lymphoma in 87.5%, follicle center cell lymphoma in 78%, chromic lymphocytic leukemia in 78%, immunocytoma in 75% and marginal zone B-cell lymphoma in 80%. CONCLUSION: The application of texture analysis is feasible in the classification of NHL.

Azure Stains↗
PubMed1991

Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates.

Comparison of published methods for the quantification of adherent cell numbers by the measurement of absorbance of bound stain indicates a wide variation in their sensitivity. This study aimed at comparing the sensitivities of five different staining procedures (Coomassie brilliant blue G in perchloric acid, Coomassie brilliant blue G in phosphoric acid, methylene blue, crystal violet, and toluidine blue) applied to three separate types of cultured fibroblasts (3T3 cells, Vero cells, and human gingival fibroblasts) at concentrations from 0.125 x 10(4) to 10 x 10(4) per well in 96-well microplates. Absorbance values of Coomassie blue-stained cells were measured in situ. Those of the remaining cells were measured after solubilization of the dye with 1% sodium dodecyl sulfate. All absorbance values were measured using an Elisa reader at 620 or 570 nm for crystal violet. The relationship between cell number and absorbance over the entire cell concentration range was best fitted with quadratic regression analysis, in contrast with the linear relationship described elsewhere. The order of sensitivity of the staining procedures was the same for each cell type: Coomassie blue in perchloric acid less than Coomassie blue in phosphoric acid less than methylene blue less than crystal violet less than toluidine blue. With the latter two stains absorbance values began to plateau at approximately 8 x 10(4) cells per well. However, staining with Coomassie blue in perchloric acid and methylene blue resulted in an almost linear relationship between cell number and absorbance over the entire concentration range tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Count↗
PubMed1997

Intraoperative identification of sentinel lymph node in patients with malignant melanoma.

We report our experience with the technique of lymphatic mapping using patent blue V dye in patients with limb malignant melanoma. The technique is based on the hypothesis that embolic metastases occur along lymphatic channels to a 'sentinel' lymph node: the draining lymph node nearest the site of the primary malignant melanoma. Patent blue V dye (0.5-1.0 ml) is injected intradermally around the site of the melanoma. Immediately the groin or axilla is opened and the blue lymphatic channels followed to the sentinel node. The node is removed and examined by both haematoxylin and eosin (H&E) and immunohistochemical staining. We have carried out this technique in 35 patients, all of whom had 'clinically assessed' stage I disease. In all 35 patients, sentinel nodes were identified, and nine were found to contain unsuspected micrometastases. Our initial evaluation of intraoperative lymphatic mapping is very promising. The technique is practicable and easy to master. If 25% of patients with cutaneous malignant melanoma who are clinically stage I have nodal disease, this has great importance not only for staging and treatment but also for all future therapeutic trials.

Adult↗
PubMed1981

Physicochemical characteristics of patent blue violet dye.

Physicochemical data for patent blue violet dye (I) are reported. The pKa for protonation of the first diethylanilino group was 2.78 +/- 0.03. The absorptivity values calculated for a 1% (w/v) solution of previously dried I at pH 7.4 were 1650, 170, and 250 at 638, 412, and 309 nm, respectively. A table of wavelength maxima and observed solution color as a function of pH and Ho and five spectra of I at certain pH and Ho values are included. The solution chemistry of I is explained, and a scheme showing its two protonated carbonium ions and its triphenylcarbinol derivative is presented. The distribution coefficients of I in n-octanol or chloroform and pH 7.4 phosphate buffer systems were 0.013 and 0.12, respectively. The approximate solubilities at 25 degrees of I in six organic solvents and the solubility analysis of I in distilled water are reported. Results of the latter analysis suggest that I forms a lyotropic mesophase in high aqueous concentrations. Compound I is poorly lipid soluble. Samples of 1.000% I in 0.9% NaCl, formulated with and without 1% (v/v) benzyl alcohol and autoclaving, varied not more than 5% from the initial I content during storage in the dark and under constant fluorescent light at 25 +/- 5 degrees for 20 months. Data from the TLC of I in several eluents indicated a high degree of purity of the dye. The half-lives for the loss of color in 5 X 10(-4)% I solutions in potassium hydroxide solutions of pH 13.7, 12.7, 11.3, and 10.0 were 1.2 hr, 17.0 hr, 9.5 days, and 180 days, respectively. The fraction of I bound to 4% (w/v) human serum albumin at 37 degrees and pH 7.4 ranged from 0.05 to 0.83, corresponding to unbound I in the postdialysis concentration range of 1.7 X 10(-4) to 2.0% (w/v). A Scatchard plot of the albumin binding data of I revealed one high-affinity binding site, K = 6235 M-1, and five low-affinity sites, with average affinity constants of 33 M-1. The data support the fact that the spectrophotometric determination of I at 639 +/- 2 nm appears to comprise a stability-indicating assay.

Chemical Phenomena↗
PubMedDate not supplied

Implementation of sentinel lymph node biopsy with blue dye outside a specialized center: can we improve quality assurance?

Implementation of sentinel lymph node biopsy (SLNB) outside a specialized center requires adequate quality control and quality assurance for optimal patient care. We examine the model of a supervising breast cancer unit in improving quality assurance in the treatment of breast cancer patients. A total of 76 SLNBs followed by level I/II axillary dissection were performed in the general hospital by using patent blue V alone. Patient selection, technical information, documentation, pathologic examination of the sentinel lymph node (SLN), and surgical training were provided by the supervising breast cancer unit. In 73 of 76 SLNBs, identification of the sentinel node was successful. The false-negative rate was 2.7% and the accuracy was 98.6%. The results of the procedure in the general hospital were comparable with the results of the supervising breast cancer unit. The model of a supervising breast cancer unit seems to accelerate surgeons' learning curve and may provide support for appropriate application of the SLNB in a wider number of nonspecialized centers.

Adult↗
PubMed1955

Evidence of differences in the desoxyribonucleoprotein complex of rapidly proliferating and non-dividing cells.

1. Quantitative cytophotometric analysis of the interphase cells of a rapidly proliferating differentiated tissue such as liver of new born rat, indicates that these cells can be separated into two groups on the basis of their staining characteristics after methanol fixation. 2. These groups are thought to correspond to two stages of interphase. The first, called "autosynthetic interphase," comprises cells which are duplicating chromosomal material in preparation for mitosis, and shows parallel increases in the methyl green and Feulgen staining of DNA and the fast green staining of histone from the diploid (2 C) to double these values (4 C). 3. The second group is designated the "heterosynthetic interphase," during which the cell ceases proliferating and functions in a manner commensurate with its state of differentiation. In this stage Feulgen staining indicates the diploid chromosomal complement, but there is a decreased capacity of the DNA to bind methyl green and of the histone to bind fast green. 4. The difference between the methyl green binding of the heterosynthetic and autosynthetic 2 C cells is due to the presence of a protein in the former which presumably inhibits staining by competing with the dye for binding sites on the DNA. The effect of this inhibition can be removed by extracting the protein, or by blocking the protein basic groups. 5. The decreased fast green staining of histone in the heterosynthetic cells can be minimized by prolonged fixation with formaldehyde. It is thought to stem either from a similar type of inhibition, or from an increased susceptibility of the histone to loss from the cell during this stage. 6. While histone inhibits methyl green staining of DNA in all cells, the differences between the staining properties of the autosynthetic and heterosynthetic interphase cells are believed to be due to another protein, whose properties appear similar to those of the chromosomal "residual protein." It is concluded that a complex of DNA and residual protein existing during the heterosynthetic interphase is dissociated during the autosynthetic interphase.

Animals↗
PubMed1989

'In situ' Edman degradation of protein(s) blotted to immobilon membranes suitable for unblocking reversible chemical modification and elimination of coomassie blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Conditions for unblocking reversible chemical modifications such as maleylation or citraconylation 'in situ' at the N-terminus of proteins after transfer of proteins to immobilon membranes from SDS-PAGE are described. Demaleylation or decitraconylation occurred at 55 degrees C in 70% formic acid (pH 1.50) during 60 min. During the unblocking reaction, Coomassie blue dye was completely removed, resulting in superior high performance liquid chromatographic separation of phenylthiohydantoin-amino acid (PTH-AA) after Edman degradation (automatic gas phase sequencer). The protein fixed on the matrix after demaleylation and removal of Coomassie blue was not degraded. The possible cleavage at the aspartyl-prolyl peptide bonds was considered, but no side reaction was observed. Furthermore, the incubation time in 70% formic acid at 55 degrees C could be reduced to 10 min in the absence of maleylation of the starting material, and this was suitable for the removal of Coomassie blue and the quantification of phenylthiolhydantoin-amino acids (PTH-AAs) by HPLC. The yield from the starting protein through SDS-PAGE, blotting, and Edman degradation to quantitative analysis of PTH-aminoacid(s) by HPLC was established.

Amino Acids↗